The goal of this research is a molecular understanding of the early steps of the Drosophila sex determination pathway as an example of an experimentally tractable developmental switch. The focus is the regulatory gene Sex-lethal, which functions at the start of a short hierarchy of genes that are regulated by sex-specific, alternative RNA splicing. Sxl produces transcripts that are spliced into a functional form in females but a non-functional form in males. Sxl is the first identified gene after the initial pathway signal, the ratio of X chromosomes to autosomes. Sxl's early response is characterized by a set of female-specific transcripts observed transiently in early embryogenesis; these are distinct from the later transcripts regulated by splicing. Regulatory genes that control this early step will be identified and characterized in order to elucidate the mechanism capable of distinguishing between one and two X chromosomes. The approach will be to devise genetic screens for suppressors of existing signal element mutations, and to screen for proteins that bind enhancers of the Sxl early promoter. Interactions between regulatory proteins and with DNA will be examined. The relevance of Drosophila to human development has been well established with the description of homeobox genes and their pattern of expression in mammalian embryos. Definition of a genetic switch such as Drosophila sex determination may likewise be expected to provide a model for human regulatory mechanisms in development.